In-vitro and in-silico anti-HSV-1 activity of a marine steroid from the jellyfish Cassiopea andromeda venom.

In this study, we investigated the potential in vitro anti-HSV-1 activities of the Cassiopea andromeda jellyfish tentacle extract (TE) and its fractions, as well as computational work on the thymidine kinase (TK) inhibitory activity of the identified secondary metabolites. The LD50, secondary metabolite identification, preparative and analytical chromatography, and in silico TK assessment were performed using the Spearman-Karber, GC-MS, silica gel column chromatography, RP-HPLC, LC-MS, and docking methods, respectively. The antiviral activity of TE and the two purified compounds Ca2 and Ca7 against HSV-1 in Vero cells was evaluated by MTT and RT-PCR assays. The LD50 (IV, mouse) values of TE, Ca2, and Ca7 were 104.0 ± 4, 5120 ± 14, and 197.0 ± 7 (µg/kg), respectively. They exhibited extremely effective antiviral activity against HSV-1. The CC50 and MNTD of TE, Ca2, and Ca7 were (125, 62.5), (25, 12.5), and (50, 3.125) µg/ml, respectively. GC-MS analysis of the tentacle extract revealed seven structurally distinct chemical compositions. Four of the seven compounds had a steroid structure. According to the docking results, all compounds showed binding affinity to the active sites of both thymidine kinase chains. Among them, the steroid compound Pregn-5-ene-3,11-dione, 17,20:20,21 bis [methylenebis(oxy)]-, cyclic 3-(1,2-ethane diyl acetal) (Ca2) exhibited the highest affinity for both enzyme chains, surpassing that of standard acyclovir. In silico data confirmed the experimental results. We conclude that the oxosteroid Ca2 may act as a potent agent against HSV-1.

Toxin metalloproteinases exert a dominant influence on pro-inflammatory response and anti-inflammatory regulation in jellyfish sting dermatitis.

Toxin metalloproteinases are the primary components responsible for various toxicities in jellyfish venom, and there is still no effective specific therapy for jellyfish stings. The comprehension of the pathogenic mechanisms underlying toxin metalloproteinases necessitates further refinement. In this study, we conducted a differential analysis of a dermatitis mouse model induced by jellyfish Nemopilema nomurai venom (NnNV) samples with varying levels of metalloproteinase activity. Through skin tissue proteomics and serum metabolomics, the predominant influence of toxin metalloproteinase activity on inflammatory response was revealed, and the signal pathway involved in its regulation was identified. In skin tissues, many membrane proteins were significantly down-regulated, which might cause tissue damage. The expression of pro-inflammatory factors was mainly regulated by PI3K-Akt signaling pathway. In serum, many fatty acid metabolites were significantly down-regulated, which might be the anti-inflammation feedback regulated by NF-κB p65 signaling pathway. These results reveal the dermatitis mechanism of toxin metalloproteinases and provide new therapeutic targets for further studies. SIGNIFICANCE: Omics is an important method to analyze the pathological mechanism and discover the key markers, which can reveal the pathological characteristics of jellyfish stings. Our research first analyzed the impact of toxin metalloproteinases on jellyfish sting dermatitis by skin proteomics and serum metabolomics. The present results suggest that inhibition of toxin metalloproteinases may be an effective treatment strategy, and provide new references for further jellyfish sting studies.

Nemopilema nomurai jellyfish venom attenuates phenotypic modulation of PDGF BB-induced vascular smooth muscle cells and κ-carrageenan-induced rat tail thrombosis.

Jellyfish venoms have long been recognized as a potentially rich source of natural bioactive compounds with pharmacological potential for the creation of innovative drugs. Our previous study demonstrated that Nemopilema nomurai jellyfish venom (NnV) has a chymotrypsin-like serine protease with fibrinolytic activity in vitro. Therefore, the present study aims to investigate the potential effect of NnV on cell migration, proliferation, and differentiation of vascular smooth muscle cells (VSMC; A7r5 cells) involved in the probable mechanism pathways. We also determined its anti-thrombotic effect through κ-carrageenan-induced Sprague-Dawley (SD) rat tail thrombus model. NnV inhibits on Platelet-derived growth factor (PDGF)-BB-stimulated A7r5 cells migration and proliferation by decreasing matrix metalloproteinase 2 (MMP-2) level and phosphorylation of ERK and Akt in a dose-dependent manner, but not p38. Furthermore, NnV regulates the phenotype transition of differentiation in PDGF-BB-stimulated A7r5 cells via ɑ-SMA and calponin in a dose-dependent manner. In an in vivo study, NnV treatment demonstrated clear anti-thrombotic activity in a dose-dependent manner, which was associated with decreased thrombus formation and length in κ-carrageenan-induced SD rat tail. These findings suggested that NnV has a novel fibrinolytic enzyme that can be used to prevent and/or treat thrombosis-related cardiovascular disorders.

Interaction of the Inhibitory Peptides ShK and HmK with the Voltage-Gated Potassium Channel KV1.3: Role of Conformational Dynamics.

Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel KV1.3 with IC50 values in the pM range and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. Nuclear magnetic resonance (NMR) relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for the KV1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK) and has implications for the design of KV1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homologue, HmK, to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the KV1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 µs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to KV1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to KV1.3 in the case of ShK. Both peptides bind to KV1.3 with Lys22 occupying the pore of the channel. Intriguingly, the more flexible peptide, ShK, binds with significantly higher affinity than HmK.

Chemical Compositions and Experimental and Computational Modeling of the Anticancer Effects of Cnidocyte Venoms of Jellyfish Cassiopea andromeda and Catostylus mosaicus on Human Adenocarcinoma A549 Cells.

Nowadays, major attention is being paid to curing different types of cancers and is focused on natural resources, including oceans and marine environments. Jellyfish are marine animals with the ability to utilize their venom in order to both feed and defend. Prior studies have displayed the anticancer capabilities of various jellyfish. Hence, we examined the anticancer features of the venom of Cassiopea andromeda and Catostylus mosaicus in an in vitro situation against the human pulmonary adenocarcinoma (A549) cancer cell line. The MTT assay demonstrated that both mentioned venoms have anti-tumoral ability in a dose-dependent manner. Western blot analysis proved that both venoms can increase some pro-apoptotic factors and reduce some anti-apoptotic molecules that lead to the inducing of apoptosis in A549 cells. GC/MS analysis demonstrated some compounds with biological effects, including anti-inflammatory, antioxidant and anti-cancer activities. Molecular docking and molecular dynamic showed the best position of each biologically active component on the different death receptors, which are involved in the process of apoptosis in A549 cells. Ultimately, this study has proven that both venoms of C. andromeda and C. mosaicus have the capability to suppress A549 cells in an in vitro condition and they might be utilized in order to design and develop brand new anticancer agents in the near future.

Purification of PaTx-II from the Venom of the Australian King Brown Snake and Characterization of Its Antimicrobial and Wound Healing Activities.

Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC50 > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.

Inhibition of Nematocyst Discharge from Pelagia noctiluca (Cnidaria: Scyphozoa)-Prevention Measures against Jellyfish Stings.

Pelagia noctiluca stings are common in Mediterranean coastal areas and, although the venom is non-lethal, they are painful. Due to its high toxicity and abundance, P. noctiluca is considered a target species for the focus of research on active ingredients to reduce the symptoms of its sting. To determine the effect of 31 substances and formulations on nematocyst discharge, we performed three tests: (1) screening of per se discharge activator solutions, (2) inhibitory test with nematocyst chemical stimulation (5% acetic acid) and (3) inhibitory test quantifying the hemolytic area. Ammonia, barium chloride, bleach, scented ammonia, carbonated cola, lemon juice, sodium chloride and papain triggered nematocyst discharge. All of them were ruled out as potential inhibitors. Butylene glycol showed a reduction in nematocyst discharge, while the formulations of 10% lidocaine in ethanol, 1.5% hydroxyacetophenone in distilled water + butylene glycol, and 3% Symsitive® in butylene glycol inhibited nematocyst discharge. These last results were subsequently correlated with a significant decrease in hemolytic area in the venom assays versus seawater, a neutral solution. The presented data represent a first step in research to develop preventive products for jellyfish stings while at the same time attempting to clarify some uncertainties about the role of various topical solutions in P. noctiluca first-aid protocols.

Discovery of a novel jellyfish venom metalloproteinase inhibitor from secondary metabolites isolated from jellyfish-derived fungus Aspergillus versicolor SmT07.

The major jellyfish stings that occur in China are caused by scyphozoan Nemopilema nomurai, whose venom exhibits significant metalloproteinase activity that contributes to the toxic effects of jellyfish envenomation. Researching effective inhibitors suppressing the metalloproteinase activity of jellyfish venom represents a new attempt to cure jellyfish envenomations. In the present study, secondary metabolites produced by the jellyfish-associated fungus Aspergillus versicolor SmT07 were isolated and evaluated for their anti-proteolytic activities. Two xanthones, sterigmatocystin (JC-01) and oxisterigmatocystin C (JC-06), and four alkaloids, cottoquinazoline A (JC-02), phenazine-1-carboxylic acid (JC-03), viridicatin (JC-04) and viridicatol (JC-05), were isolated and identified. Only phenazine-1-carboxylic acid (PCA) showed significant anti-proteolytic activity of jellyfish venom assayed on azocasein, and the IC50 value was 2.16 mM. PCA also significantly inhibited fibrinogenolytic activity, protecting the Bß chain of fibrinogen from degradation when preincubated with jellyfish venom at a ratio of >1:0.6 (PCA:venom, w/w). Molecular docking with several well-characterized snake venom metalloproteinases suggested the venom metalloproteinases inhibitory property of PCA by forming complex interactions with the active site via hydrogen bonds, π-π stacking and salt bridges, which was distinct from the binding mode of batimastat. The present study represents the first study identifying natural jellyfish venom metalloproteinase inhibitors from marine natural products, which may provide an alternative to develop therapeutic agents for treating jellyfish envenomations.

Sea anemone Bartholomea annulata venom inhibits voltage-gated Na+ channels and activates GABAA receptors from mammals.

Toxin production in nematocysts by Cnidaria phylum represents an important source of bioactive compounds. Using electrophysiology and, heterologous expression of mammalian ion channels in the Xenopus oocyte membrane, we identified two main effects produced by the sea anemone Bartholomea annulata venom. Nematocysts isolation and controlled discharge of their content, revealed that venom had potent effects on both voltage-dependent Na+ (Nav) channels and GABA type A channel receptors (GABAAR), two essential proteins in central nervous system signaling. Unlike many others sea anemone toxins, which slow the inactivation rate of Nav channels, B. annulata venom potently inhibited the neuronal action potential and the Na+ currents generated by distinct Nav channels opening, including human TTX-sensitive (hNav1.6) and TTX-insensitive Nav channels (hNav1.5). A second effect of B. annulata venom was an agonistic action on GABAAR that activated distinct receptors conformed by either α1ß2γ2, α3ß2γ1 or, ρ1 homomeric receptors. Since GABA was detected in venom samples by ELISA assay at low nanomolar range, it was excluded that GABA from nematocysts directly activated the GABAARs. This revealed that substances in B. annulata nematocysts generated at least two potent and novel effects on mammalian ion channels that are crucial for nervous system signaling.

Nomad Jellyfish Rhopilema nomadica Venom Induces Apoptotic Cell Death and Cell Cycle Arrest in Human Hepatocellular Carcinoma HepG2 Cells.

Jellyfish venom is a rich source of bioactive proteins and peptides with various biological activities including antioxidant, antimicrobial and antitumor effects. However, the anti-proliferative activity of the crude extract of Rhopilema nomadica jellyfish venom has not been examined yet. The present study aimed at the investigation of the in vitro effect of R. nomadica venom on liver cancer cells (HepG2), breast cancer cells (MDA-MB231), human normal fibroblast (HFB4), and human normal lung cells (WI-38) proliferation by using MTT assay. The apoptotic cell death in HepG2 cells was investigated using Annexin V-FITC/PI double staining-based flow cytometry analysis, western blot analysis, and DNA fragmentation assays. R. nomadica venom displayed significant dose-dependent cytotoxicity on HepG2 cells after 48 h of treatment with IC50 value of 50 µg/mL and higher toxicity (3:5-fold change) against MDA-MB231, HFB4, and WI-38 cells. R. nomadica venom showed a prominent increase of apoptosis as revealed by cell cycle arrest at G2/M phase, upregulation of p53, BAX, and caspase-3 proteins, and the down-regulation of anti-apoptotic Bcl-2 protein and DNA fragmentation. These findings suggest that R. nomadica venom induces apoptosis in hepatocellular carcinoma cells. To the best of the authors' knowledge, this is the first scientific evidence demonstrating the induction of apoptosis and cell cycle arrest of R. nomadica jellyfish venom.

Charge substitutions at the voltage-sensing module of domain III enhance actions of site-3 and site-4 toxins on an insect sodium channel.

Scorpion α-toxins bind at the pharmacologically-defined site-3 on the sodium channel and inhibit channel inactivation by preventing the outward movement of the voltage sensor in domain IV (IVS4), whereas scorpion ß-toxins bind at site-4 on the sodium channel and enhance channel activation by trapping the voltage sensor of domain II (IIS4) in its outward position. However, limited information is available on the role of the voltage-sensing modules (VSM, comprising S1-S4) of domains I and III in toxin actions. We have previously shown that charge reversing substitutions of the innermost positively-charged residues in IIIS4 (R4E, R5E) increase the activity of an insect-selective site-4 scorpion toxin, Lqh-dprIT3-c, on BgNav1-1a, a cockroach sodium channel. Here we show that substitutions R4E and R5E in IIIS4 also increase the activity of two site-3 toxins, LqhαIT from Leiurusquinquestriatus hebraeus and insect-selective Av3 from Anemonia viridis. Furthermore, charge reversal of either of two conserved negatively-charged residues, D1K and E2K, in IIIS2 also increase the action of the site-3 and site-4 toxins. Homology modeling suggests that S2-D1 and S2-E2 interact with S4-R4 and S4-R5 in the VSM of domain III (III-VSM), respectively, in the activated state of the channel. However, charge swapping between S2-D1 and S4-R4 had no compensatory effects on gating or toxin actions, suggesting that charged residue interactions are complex. Collectively, our results highlight the involvement of III-VSM in the actions of both site 3 and site 4 toxins, suggesting that charge reversing substitutions in III-VSM allosterically facilitate IIS4 or IVS4 voltage sensor trapping by these toxins.

Field Experiment Effect on Citrus Spider Mite Panonychus citri of Venom from Jellyfish Nemopilema nomurai: The Potential Use of Jellyfish in Agriculture.

Jellyfish are rich in resources and widely distributed along coastal areas. As a potential approach to respond to jellyfish blooms, the use of jellyfish-derived products is increasing. The citrus spider mite (Panonychus citri) is one of the key citrus pests, negatively impacting the quality and quantity of oranges. Due to the resistance and residue of chemical acaricides, it is important to seek natural substitutes that are environmentally friendly. The field efficacy of the venom from the jellyfish Nemopilema nomurai against P. citri was assayed in a citrus garden. The frozen N. nomurai tentacles were sonicated in different buffers to isolate the venom. The venom isolated by PBS buffer (10 mM, pH 6.0) had the strongest acaricidal activity of the four samples, and the corrected field efficacy 7 days after treatment was up to 95.21%. This study demonstrated that jellyfish has potential use in agriculture.

Spontaneous myogenic fasciculation associated with the lengthening of cardiac muscle in response to static preloading.

Force enhancement is one kind of myogenic spontaneous fasciculation in lengthening preload striated muscles. In cardiac muscle, the role of this biomechanical event is not well established. The physiological passive property is an essential part for maintaining normal diastole in the heart. In excessive preload heart, force enhancement relative erratic passive properties may cause muscle decompensating, implicate in the development of diastolic dysfunction. In this study, the force enhancement occurrence in mouse cardiac papillary muscle was evaluated by a microstepping stretch method. The intracellular Ca2+ redistribution during occurrence of force enhancement was monitored in real-time by a Flou-3 (2 mM) indicator. The force enhancement amplitude, the enhancement of the prolongation time, and the tension-time integral were analyzed by myography. The results indicated that the force enhancement occurred immediately after active stretching and was rapidly enhanced during sustained static stretch. The presence of the force and the increase in the amplitude synchronized with the acquisition and immediate transfer of Ca2+ to adjacent fibres. In highly preloaded fibres, the enhancement exceeded the maximum passive tension (from 4.49 ± 0.43 N/mm2 to 6.20 ± 0.51 N/mm2). The occurrence of force enhancement were unstable in each static stretch. The increased enhancement amplitude combined with the reduced prolongation time to induce a reduction in the tension-time integral. We concluded that intracellular Ca2+-synchronized force enhancement is one kind of interruption event in excessive preload cardiac muscle. During the cardiac muscle in its passive relaxation period, the occurrence of this interruption affected the rhythmic stability of the cardiac relaxation cycle.

Arrhythmogenic and antiarrhythmic actions of late sustained sodium current in the adult human heart.

Late sodium current (late INa) inhibition has been proposed to suppress the incidence of arrhythmias generated by pathological states or induced by drugs. However, the role of late INa in the human heart is still poorly understood. We therefore investigated the role of this conductance in arrhythmias using adult primary cardiomyocytes and tissues from donor hearts. Potentiation of late INa with ATX-II (anemonia sulcata toxin II) and E-4031 (selective blocker of the hERG channel) slowed the kinetics of action potential repolarization, impaired Ca2+ homeostasis, increased contractility, and increased the manifestation of arrhythmia markers. These effects could be reversed by late INa inhibitors, ranolazine and GS-967. We also report that atrial tissues from donor hearts affected by atrial fibrillation exhibit arrhythmia markers in the absence of drug treatment and inhibition of late INa with GS-967 leads to a significant reduction in arrhythmic behaviour. These findings reveal a critical role for the late INa in cardiac arrhythmias and suggest that inhibition of this conductance could provide an effective therapeutic strategy. Finally, this study highlights the utility of human ex-vivo heart models for advancing cardiac translational sciences.

Anti-Inflammatory and Analgesic Effects of TRPV1 Polypeptide Modulator APHC3 in Models of Osteo- and Rheumatoid Arthritis.

Arthritis is a widespread inflammatory disease associated with progressive articular surface degradation, ongoing pain, and hyperalgesia causing the development of functional limitations and disability. TRPV1 channel is one of the high-potential targets for the treatment of inflammatory diseases. Polypeptide APHC3 from sea anemone Heteractis crispa is a mode-selective TRPV1 antagonist that causes mild hypothermia and shows significant anti-inflammatory and analgesic activity in different models of pain. We evaluated the anti-inflammatory properties of APHC3 in models of monosodium iodoacetate (MIA)-induced osteoarthritis and complete Freund's adjuvant (CFA)-induced rheumatoid monoarthritis in comparison with commonly used non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, and meloxicam. Subcutaneous administration of APHC3 (0.1 mg/kg) significantly reversed joint swelling, disability, grip strength impairment, and thermal and mechanical hypersensitivity. The effect of APHC3 was equal to or better than that of reference NSAIDs. Protracted treatment with APHC3 decreased IL-1b concentration in synovial fluid, reduced inflammatory changes in joints, and prevented the progression of cartilage degradation. Therefore, polypeptide APHC3 has the potential to be an analgesic and anti-inflammatory substance for the alleviation of arthritis symptoms.

Efficient Prevention of Marine Biofilm Formation Employing a Surface-Grafted Repellent Marine Peptide.

Creation of surfaces resistant to the formation of microbial biofilms via biomimicry has been heralded as a promising strategy to protect a range of different materials ranging from boat hulls to medical devices and surgical instruments. In our current study, we describe the successful transfer of a highly effective natural marine biofilm inhibitor to the 2D surface format. A series of cyclic peptides inspired by the natural equinatoxin II protein produced by Beadlet anemone (Actinia equine) have been evaluated for their ability to inhibit the formation of a mixed marine microbial consortium on polyamide reverse osmosis membranes. In solution, the peptides are shown to effectively inhibit settlement and biofilm formation in a nontoxic manner down to 1 nM concentrations. In addition, our study also illustrates how the peptides can be applied to disperse already established biofilms. Attachment of a hydrophobic palmitic acid tail generates a peptide suited for strong noncovalent surface interactions and allows the generation of stable noncovalent coatings. These adsorbed peptides remain attached to the surface at significant shear stress and also remain active, effectively preventing the biofilm formation over 24 h. Finally, the covalent attachment of the peptides to an acrylate surface was also evaluated and the prepared coatings display a remarkable ability to prevent surface colonization at surface loadings of 55 ng/cm2 over 48 h. The ability to retain the nontoxic antibiofilm activity, documented in solution, in the covalent 2D-format is unprecedented, and this natural peptide motif displays high potential in several material application areas.

Impact of pacing frequency in amiodarone interaction with cardiomyocytes near physiological temperature in health and disease conditions.

Long QT syndrome type 3 (LQT-3) is a disease related to abnormal cardiac sodium channel function (Nav 1.5), usually due to augmented late sodium current (INaL ), and may lead to ventricular fibrillation. Amiodarone is approved for ventricular fibrillation. Thus, we investigated whether pacing frequency impacts the ability of amiodarone to reverse the arrhythmic phenotype observed in LQT-3. Anemone neurotoxin 2 (ATX-II, here named only ATX) was used to enhance INaL in mice left ventricular myocytes (LVM). A video detector system monitored sarcomere shortening. At 1 Hz, amiodarone attenuated sarcomere shortening only at 10 µmol/L; at 3 and 5 Hz, 0.1 and 1 µmol/L amiodarone also reduced sarcomere shortening. However, no effect of amiodarone was observed on time to 50% of sarcomere contraction and relaxation. In LVM exposed to ATX (10 nmol/L), an arrhythmic phenotype was observed, and it was more severe when cells were paced at 1 Hz. Amiodarone failed to reverse the ATX induced phenotype at different pacing frequencies. Thus, our results suggest that amiodarone's ability to reverse arrhythmias induced by augmentation of INaL is limited. These findings suggest further experimentation will be required to clarify whether a clinical effect can be ascribed to an effect of amiodarone on other ion channels in LQT-3.

In Silico Laboratory Experiments Using Statistical Model Checking: A New Model of the Palytoxin-Induced Pump Channel as Case Study.

Studying biological systems is a difficult but important task. Traditional methods include laboratory experimentation and computer simulations. However, often researchers need to explore important but potentially rare events that are not easily observed or simulated. We use UPPAAL-SMC, a formal verification tool to support a methodology that allows us to model biological systems, specify events and conditions that we want to analyze, and to explore system executions using controlled simulations. We also describe an efficient way to reproduce laboratory experiments in silico. Unlike traditional simulations, we are able to guide the experiment to explore special events and conditions by expressing these conditions in temporal logic formulas. We have applied this methodology to create a more detailed model of Palytoxin-induced Na +/K + pump channels than was previously possible. Moreover, we have reproduced experimental protocols and their associated electrophysiological recordings, which has not been done in previous works. As a consequence, we have been able to propose a new diprotomeric model for the PTX-pump complex and study its behaviour. The use of our methodology has enabled us to reduce the effort and time to perform this research. It can be used to model and analyze other complex biological systems, potentially increasing the productivity of such studies.

Sea Anemone Heteractis crispa Actinoporin Demonstrates In Vitro Anticancer Activities and Prevents HT-29 Colorectal Cancer Cell Migration.

Actinoporins are the most abundant group of sea anemone cytolytic toxins. Their membranolytic activity is of high interest for the development of novel anticancer drugs. However, to date the activity of actinoporins in malignant cells has been poorly studied. Here, we report on recombinant analog of Hct-S3 (rHct-S3), belonging to the combinatory library of Heteractis crispa actinoporins. rHct-S3 exhibited cytotoxic activity against breast MDA-MB-231 (IC50 = 7.3 µM), colorectal HT-29 (IC50 = 6.8 µM), and melanoma SK-MEL-28 (IC50 = 8.3 µM) cancer cells. The actinoporin effectively prevented epidermal growth factor -induced neoplastic transformation of JB6 Cl41 cells by 34% ± 0.2 and decreased colony formation of HT-29 cells by 47% ± 0.9, MDA-MB-231 cells by 37% ± 1.2, and SK-MEL-28 cells by 34% ± 3.6. Moreover, rHct-S3 decreased proliferation and suppressed migration of colorectal carcinoma cells by 31% ± 5.0 and 99% ± 6.4, respectively. The potent anti-migratory activity was proposed to mediate by decreased matrix metalloproteinases-2 and -9 expression. In addition, rHct-S3 induced programmed cell death by cleavage of caspase-3 and poly (ADP-ribose) polymerase, as well as regulation of Bax and Bcl-2. Our results indicate rHct-S3 to be a promising anticancer drug with a high anti-migratory potential.

New Sea Anemone Toxin RTX-VI Selectively Modulates Voltage-Gated Sodium Channels.

A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (NaV) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the NaV channels. Electrophysiological screening of RTV-VI on NaV channel subtypes showed its selective interaction with the central nervous system (NaV1.2, NaV1.6) and insect (BgNaV1, VdNaV1) sodium channels.